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Vol. 3, 2055-2061. November 1997 Clinical Cancer Research 2055
DNA Repair and Cellular Resistance to Alkylating Agents in
Chronic Lymphocytic Leukemia’
Mark R. Muller,2 Claudia Buschfort,
J#{252}rgenThomale, Carmen Lensing,
Manfred F. Rajewsky, and Siegfried SeeberDepartment of Internal Medicine (Cancer Research) [M. R. M.. C. L.,
S. 5.1 and Institute of Cell Biology (Cancer Research) [J. T.. C. B.,M. F. R.], West German Cancer Center Essen, University of EssenMedical School. D-45122 Essen, Germany
ABSTRACT
The time course of the formation and persistence of
repair-induced DNA lesions such as single-strand breaks
(SSBs) were determined in isolated lymphocytes derived
from 32 patients with chronic lymphocytic leukemia (CLL)
using the single-cell gel electrophoresis (SCGE, “comet”)
assay. After pulse-exposure to N-ethyl-N-nitrosourea
(EtNU), the initial amount of SSBs (t0 SCGE values) and the
time periods required to reduce DNA damage by 50% (t50%
SCGE values) were determined in nuclear DNA of individual
cells. The 10 SCGE and t50% SCGE values varied interindividu-
ally between CLL specimens by factors of 16.6 and 8.2,
respectively. Regarding cell-to-cell variation, no major sub-
populations with significantly different DNA repair capaci-
ties were observed in cell specimens from a given patient. In
addition, a monoclonal antibody-based immunocytological
assay was used to determine the elimination kinetics for the
cytotoxic alkylation product 06-ethylguanine from nuclear
DNA. A strong correlation was observed between the rela-
tive times for SSB repair and the elimination of 06-ethyb-
guanine from nuclear DNA. Because SCGE and immunocy-
tologicab assay measure different steps of DNA repair, this
observation suggests coordinated regulation of the respec-
tive repair pathways. With regard to chemosensitivity pro-
files, a “fast” repair phenotype corresponded to enhanced
in vitro resistance to EtNU, 1,3-bis(2-chboroethyl).1-
nitrosourea, or chlorambucil. Accelerated SSB repair and
pronounced in vitro resistance to chborambucil, 1,3-bis(2-
chboroethyl)-1-nitrosourea, and EtNU were found in lym-
phocytes from CLL patients nonresponsive to chemotherapy
with alkylating agents. Distinct DNA repair processes thus
mediate resistance to alkylating agents in CLL lymphocytes.
Received 3/28/97; revised 7/I 1/97; accepted 7/I 1/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
I This work was supported by Grant W769/94/MU2 from Dr. Mildred
Scheel Stiftung.2 To whom requests for reprints should be addressed, at Innere Klinikund Poliklinik (Tumorforschung), Westdeutsches Tumorzentrum Essen.Universithtsklinikum Essen, Hufelandstrasse 55, D-45122 Essen,
Bargteheide, Germany). In case of the SCGE, the amount of
DNA damage in single cells was determined by measuring
the total area of stained nuclear DNA and the fluorescence
intensity by image analysis. Comet sizes were defined as the
relative increase of fluorescent area of EtNU-treated lympho-
cytes compared to the area of nuclei of untreated control cells
from the same donor in the same experiment. For ICA, signal
intensities of 06-EtGua immunostaining and DNA staining
were expressed as the integrated values (average signal X
number of selected pixels) for each nucleus. 06-EtGua sig-
nals were corrected for nuclear DNA content.
MTT Assay. For the tetrazolium dye (MTT) assay, cells
were seeded into 96-well microtiter plates using 200 p.1/well of
a cell suspension containing 106 lymphocytes/ml of RPMI 1640
supplemented with 10% FCS. Dissolved drugs were added in 20
p.1 of PBS. On day 4, 20 p.1 of a solution of S mg MTT/ml of
PBS were added to each well, and the plates were further
incubated for 5 h. Thereafter, the plates were centrifuged for 10
mm at 100 X g. The medium was removed from each well, 200
Clinical Cancer Research 2057
Fig. 1 “Comet” formation in a CLL lymphocyte after exposure toEtNU for 20 mm (A) compared with nuclear fluorescence from an
untreated CLL lymphocyte as a control (B). The labile sites in DNA dueto repair processes appear as a comet attached to the nucleus. Theamount of DNA damage in single cells was determined by measuring
the total area of stained nuclear DNA and the fluorescence intensity byimage analysis. Comet sizes were defined as the relative increase of
fluorescence area of EtNU-treated lymphocytes compared to the area of
nuclei of untreated control cells.TIME AFTER ETNU PULSE (h)
Fig. 2 Kinetics of comet formation and disappearance (top) and of the
elimination of 06-EtGua from nuclear DNA (bottom) in two specimens
of CLL lymphocytes after in vitro pulse exposure to EtNU. Curves were
obtained from two CLL patients who were either sensitive (#{149})orresistant (0) to treatment with alkylating agents. In case of SCGE, the
area of stained nuclear DNA (mean of 150 cells; coefficient of variation,15%) is given as the increase relative to untreated control cells. Regard-
ing ICA, mean values of relative nuclear fluorescence signals of 100cells/time point are plotted (coefficient of variation. 25%). The t3�.3
values were determined graphically in all plots. lnterindividual variation
was not only observed for t5� values but also for the form of the curves
that do not follow a first-order kinetic (20, 33).
Fig. 6 Repair time for SSB in the nuclear DNA of CLL lymphocytesin relation to treatment outcome. The t50� SCGE repair values were
determined in lymphocytes obtained from CLL patients who were eitheruntreated, sensitive, or resistant to treatment with alkylating agents.Horizontal lines, means of the distributions.
Clinical Cancer Research 2059
25U�
200
1 50
100� #{149}#{149}#{149}.#{149}I�
�, ,#{149} S50 #{149}#{149} S
0#{149} I I I I I I
0 10 20 30 40 50 60 70
t50% values (mm)
Fig. 5 Correlation between chemosensitivity in vitro and DNA repair time for SSBs in CLL lymphocytes. The Y-axes display ID��s for EtNU.
BCNU, and CLB, respectively, and the X-axes the time interval required for the reduction of initial comet formation by 50% (t5�% �4-� values). The
Spearman rank correlation coefficients (rfl) for DNA repair time and cytotoxicity were r� = -0.71 for EtNU, r� = -0.54 for BCNU, and r� = -0.69
for CLB, respectively (P < 0.001).
SCGE and ICA simultaneously (Fig. 4). A positive correla-
tion was observed between the t50% values for both the
elimination of 06-EtGua from nuclear DNA and the repair of
SSBs (r = 0.98, P < 0.015).
Table I Chemosensitivity of isolated lymphocytes derived from
untreated (U), treated sensitive (TS), and treated resistant (TR)
CLL patients
The ID50s were determined after a 4-day exposure to alkylatingagents using the MiT assay.
unrelated to clinical status (P > 0.05). Table 1 shows the ID50s
for alkylating agents determined in CLL lymphocytes in relation
to clinical status. The mean ID50s for EtNU, BCNU, and CLB
were significantly elevated in lymphocytes from CLL patients
resistant to chemotherapy with CLB in comparison to untreated
or treated sensitive patients.
DISCUSSION
The SCGE comet assay was applied to evaluate in vitro the
efficiency of DNA excision repair in individual CLL lympho-
cytes in relation to DNA monoadduct elimination, chemosensi-
tivity to alkylating agents, and to clinical status. EtNU-induced
DNA monoadducts, such as 06-EtGua, are partly removed from
DNA by the repair protein AT in a one-step process (3). Fur-
thermore, 06-AlkGua is a substrate for mismatch repair pro-
cesses, and excision repair also contributes to the elimination of
06-AlkGua from nuclear DNA (7-9). During these processes,
SSB in DNA result from the incision by endonucleases as repair
intermediates. Following pulse-exposure to EtNU, the initial
numbers of SSBs varied considerably between CLL specimens.
We have shown previously that this variation is not due to
different levels of primary adduct formation but rather is related
to different efficiencies of early steps in DNA repair (33). For
example, EtNU failed to induce SSBs in vitro prior to the
addition of DNA repair proteins. Widely varying time intervals
were required for the disappearance of repair-induced SSBs, as
observed among specimens from different CLL patients. This
may reflect large interindividuab differences in the concentra-
tions/activities of DNA repair proteins in human cells, as re-
ported previously (17-20). However, no obvious relationship
was found between the initial comet size after EtNU (t0 scGE)’
used as an end point for DNA damage in most studies (27),
and #{231} S(’(iI values or any other parameter tested. One explana-
tion for this lack of correlation is that the number of SSBs
measured at a given time point after drug exposure is dependent
on several factors, such as the activity of early steps in DNA
repair (glycosybases and endonucleases) and the efficiency of
the downstream processing of DNA lesions by, e.g. , DNA
polymerases/-bigases during the incubation period.
DNA repair phenotypes are heterogeneous not only be-
tween individuals but also among different cell types (34, 35).
To monitor the DNA repair capacity of tumor cells from cancer
patients. reliable, quantitative assays are required that allow the
selective analysis of individual cells in heterogeneous biopsy
material. The present approach does not permit the measurement
of DNA repair efficiency in a given cell as a function of time;
however, it enabled us to evaluate cell-to-cell variation among a
small number of cells by SCGE and ICA. Nevertheless, detect-
able subpopulations of CLL lymphocytes with DNA repair
phenotypes significantly different from the average value for the
total cell population were not observed among the lymphocyte
specimens examined in this study.
ICA measures the efficiency of early steps of 06-EtGua
repair including those effected by AT or excision repair pro-
teins. Later stages of DNA damage processing, such as gap
filling and rejoining of SSB during excision repair, are moni-
tored by SCGE. It is, therefore, interesting to note that the
relative repair rates determined by these functional assays coy-
ering different areas of the DNA repair network were correlated.
This correlation suggests at beast partial coordination of differ-
ent rate-limiting repair components. It remains to be determined
whether this reflects the predominant action of a certain repair
pathway measured by both assays or coregulation of key com-
ponents of distinct repair pathways. The latter possibility is
supported by the observed cross-resistance of CLL lymphocytes
to structurally unrelated alkylating agents inducing different
patterns of DNA adducts.
In cell lines, an inverse relationship has been reported
previously between the repair capacity of tumor cell lines for
SSB or AT activity and the chemosensitivity to alkylating
agents (36, 37). In the present study, increased in vitro chemore-
sistance to alkylating agents was observed in CLL lymphocytes
exhibiting a fast DNA repair phenotype. This suggests the
clinical importance of DNA repair in mediating cellular resist-
ance to alkylating agents in human leukemic cells. It is not yet
established whether the observed broad range of interindividual
DNA repair capacities translates into clinical responsiveness to
alkylating drugs. We have shown previously that CLL lympho-
cytes of chemotherapy-resistant patients displayed higher rates
of adduct elimination in comparison to CLL lymphocytes of
responsive patients (19). The present study has complemented
this observation by the finding that CLL lymphocytes from
nonresponsive patients exhibited faster processing of secondary,
repair-induced DNA lesions, such as SSBs, compared to control
cells. Both observations underline the clinical significance of
DNA repair as an important mechanism of resistance to alky-
lating agents in leukemic cells. Thus far, the number of speci-
mens studied is relatively small. Future studies should encom-
pass a larger number of patients to exclude biased selection. In
any event, use of functional assays such as SCGE and ICA will
provide a means to monitor DNA repair in cancer patients to
facilitate the rational design of chemotherapeutic regimens.
ACKNOWLEDGMENTS
We thank Bettina Baack for excellent technical assistance.
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